The ability to measure a wide variety of physiologically active compounds, both naturally occurring and synthetic, has become of increasing importance, both as an adjunct to diagnosis and to therapy. While for the most part, assays of physiological fluids and drugs have required clinical laboratory determinations, there is an increasing awareness of the importance of being able to carry out assay determinations in the physician's office or in the home. To be able to perform an assay in a physician's office or home requires that the assay have a simple protocol and be relatively free of sensitivity to small changes in the conditions under which the assay is carried out.
A wide range of disposable assay devices has been developed for use either in analytical laboratories or in physicians' offices or homes. These devices, because they are used by inexperienced operators, should be simple to operate and should incorporate all the reagents necessary for the test to be conducted.
One analyte of importance is cholesterol. There is a clearly established relationship between total blood cholesterol (mainly the LDL fraction) and coronary artery disease (J.A.M.A. 253: 2080-2086, 1985). New guidelines have been established for adults to identify risk groups associated with blood cholesterol levels. Since cholesterol levels can be controlled by both diet and cholesterol-lowering medications, it is useful for those individuals at risk to be able to monitor their own cholesterol at home in order to reduce the potential for heart disease. The measurement of other naturally occurring compounds of physiological importance, such as glucose, lipoproteins, etc., as well as synthetic drugs, is also of great interest. For example, therapeutics, drugs of abuse, iodothyronines, alcohol, cytokines, as well as numerous other chemical analytes could be monitored. Also of interest are microorganisms, .beta.-HCG for ectopic births, antibodies associated with disease, and the like.
Many of the most commonly used assays in disposable assay devices require an incubation step, such as requiring enzymes to act on the sample, such as for determinations of cholesterol, glucose, uric acid, and the like. Additionally, enzymes are often used as labels in immunoassays. In a conventional enzyme immunoassay, an enzyme is covalently conjugated with one component of a specifically binding antigen-antibody pair, and the resulting enzyme conjugate is reacted with a substrate to produce a signal which is detected and measured. The signal generated by the enzyme, in either the conventional chemical assay or the immunoassay, may be a color change, and the color change may be detected with the naked eye or by a spectrophotometric technique.
Many of the disposable assay devices currently in use include one or more reagent zones comprising layers incorporated with assay reagents. Among the problems encountered in use of these devices is the premature interaction or migration of these reagents, either during the manufacturing process or upon introduction of the sample to the device. Both enzymatic and chemical reactions often require incubation steps. One of the challenges in designing a truly "one-step" disposable device is to provide a means to delay the fluid flow in order to allow for proper incubation periods. This is particularly challenging for non-instrumented disposable analytical devices.
Ideally, a disposable assay device should include a means to delay the flow of the sample through the device for a predetermined time to permit incubation of the sample with the reagents or indicators present in a particular region of the device. After the incubation period, which is generally on the order of a few minutes or less, the sample then flows to the next region of the device for further processing.
An ideal flow delay means should work like a valve, with a "closed" and an "open" state. When the state is "closed", the fluid flow should stop, and when the state switches to "open", the fluid should flow through the flow-delay valve with little or no restriction, and the flow rate of the fluid through the device should be unchanged.
A wide range of disposable analytical devices has been developed which include means to control flow of fluids therethrough. However, none of these previously developed devices has a flow-delay means with a valve-like effect on the flow of fluids.
Deutsch et al., in U.S. Pat. No. 4,522,923, disclose a test device comprising a container having at least two water-soluble barriers between at least three superposed chambers. Upon introduction of an aqueous biological sample to be tested into the topmost chamber, the sample will successively mix with the contents of the chambers. The contact time in each chamber is a function of the water solubility of the barriers.
Ebersole, in U.S. Pat. No. 4,522,786, discloses a multilayer test device comprising at least two liquid permeable functional layers superposed upon one another, the layers in liquid communication, with a barrier layer separating the layers. The barrier layer is a chemically inert, liquid insoluble, foraminous septum, the foramina of which are filled with a thermally sensitive material which is liquid impermeable at assay temperatures but capable of melting when heated to provide rapid liquid communication between the function layers. This melted material then travels through to the next layer with the test liquid.
Jones, in U.S. Pat. No. 5,213,965, discloses an assay device for measuring high density lipoprotein or cholesterol in a fluid sample which contains other lipoproteins. Sieving materials chromatographically separate aggregated from non-aggregated materials in the sample as the sample flows through the matrix. A reagent reservoir slowly releases a precipitating agent into the matrix by formulating the precipitating agent with a binder for slow dissolution on contact with a sample. There is no provision for timing the delay of the sample within a region.
Vonk, in U.S. Pat. No. 5,185,127, discloses a device for assaying an analyte comprising an enclosure and a filter stack. Flow control is provided by a hydrophilic membrane which contains a binder for an analyte. This membrane is impervious to the passage of an aqueous liquid until activated by a wetting agent.
Johnston et al., in U.S. Pat. No. 4,038,485, disclose a test composition for detecting a component in a sample which comprises a reactant system which, upon contact with the sample, interacts with the component to produce a detectable response, as well as an inhibitor system which, upon contact with the sample, prevents the reactant system from interacting with the component after lapse of a predetermined time.
Amano et al., in U.S. Pat. No. 4,889,797, disclose a dry analytical element for assaying enzyme activity in a liquid comprising a support having provided thereon at least a porous liquid-spreading layer composed of fibers which do not absorb water. Here, flow control is directed to decreasing spreading of the liquid within a layer rather than through a layer.
Deneke et al., in U.S. Pat. No. 4,876,076, disclose an assay device comprising a first carrier layer having applied thereto a liquid absorbing layer and a separate second movable carrier having applied thereto a dissolvable reagent-containing layer which is not in initial contact with the first carrier layer. The reagent-containing layer is dissolved by contact with a liquid contained in the liquid absorbing layer, and the first carrier layer is positioned in the device to permit contact between the liquid absorbing layer and the reagent-containing layer by applying pressure to one of the carrier layers. Flow of sample from one layer to another is controlled by bringing the two carrier layers into contact with each other so that the sample is transferred. This type of flow control is also shown in Ramel et al., U.S. Pat. No. 4,959,324, in which a flow path is completed by moving a sample receiving pad into a gap between two assay strips.
Woodrum, in U.S. Pat. No. 4,959,305, discloses a multizone test device for immunoassays in which assay reagents are reversibly immobilized within the various layers of the device. The binding interactions within the device depend upon the interactive properties of and between the assay reagents and the matrix comprising the incorporating layer of the reversibly immobilized assay reagents, and the disruptive properties of the liquid test sample necessary to disrupt the particular reversible binding interaction between the assay reagents and the matrix to release and render useable the reagents in an analytically effective amount within the device.
Columbus, in U.S. Pat. No. 4,549,952, discloses a liquid transport device having means for increasing the viscosity of the liquid when the liquid flows past at least one surface of the device. Control of flow is achieved strictly through viscosity increase.
Hydration and expansion of a compressed foam switching element permit automatic timed sequential delivery of multiple reagents in a device disclosed by Bruce et al. in Analytical Chemical Acta 249 (1991), 263-269. This technique is not well-suited for devices that require no diluent and which are sensitive to the total volume of sample fluid required.
Hillmann et al., in U.S. Pat. No. 4,963,498, disclose methods for using specific binding pair members which result in agglutination formation. The resulting agglutinated particles may provide for changes in flow rate.
Interrupting capillary flow of liquid between two pieces of bibulous material using a liquid expandable material is shown in Kurn et al., U.S. Pat. No. 5,104,812.
Physical barriers to reduce the flow rate in a zone of a liquid transport zone are shown in Columbus, U.S. Pat. No. 4,310,399; Columbus, U.S. Pat. No. 4,618,476; and Grenner et al., in U.S. Pat. No. 5,051,237.
Liquid flow through a filter can also be controlled by reactions between the sample and a component of the filter, cf. U.S. Pat. No. 5,217,905, to Marchand et al. Similarly, Tanaka et al., in U.S. Pat. No. 4,966,784, disclose inhibiting migration of a water-soluble indicator in a reagent layer in an assay device by using a particular organic solvent.
Liotta, in U.S. Pat. No. 3,723,064, discloses a layered testing device including a first porous layer impregnated with a reagent system which reacts with the analyte to produce an end product. A membrane having plural regions with differing permeabilities is adjacent to the first porous layer. The permeability differences are obtained either by impregnating the regions with different concentrations of a chemical reactive with the end product, or by varying the pore size in the regions.
Engelmann, in U.S. Pat. No. 4,738,823, discloses a test strip which has a preselected capacity for absorbing sample. In this case, however, the amount of sample applied to the test strip is metered, rather than the amount of sample fluid that is retained for a predetermined time within a selected area of a test device.
Other workers have coated filters with a variety of coatings to alter conditions within the filter, none of which provides a valve-like action to control the flow of fluid: Nagatomo et al., U.S. Pat. No. 4,587,102.
A layer for controlling diffusion rate of sample through the device can be provided by varying the formulation ratio of a hydrophobic polymer and a hydrophilic polymer which constitute the layer, as shown by Koyama et al., U.S. Pat. No. 4,615,983. In a similar fashion, Rothe et al., in U.S. Pat. No. 4,587,099, disclose a test strip which includes a slowly absorbent layer for sucking up a fluid sample. This layer is slowly absorbent rather than a layer which retains a liquid for a predetermined period of time.
A flow-delaying polymer on an immunoassay filter is shown in Nelson, U.S. Pat. No. 4,923,680.
Blood clotting has been used to inhibit sample flow through a track in Lucas, U.S. Pat. No. 5,207,988.
Leeder et al., in U.S. Pat. Nos. 4,837,395 and 5,089,383, disclose a heterogeneous immunoassay in which the production of the signal is temporarily delayed by using an inhibitor which can be an alternate substrate for the enzyme signal or a compound which reacts with the product of the enzyme and its substrate.
Terminiello et al., in U.S. Pat. No. 4,774,192, disclose a dry chemistry reagent system comprising a porous membrane of essentially uniform composition which has a porosity gradient from one planar surface thereof to the other.
Kuroda et al., in U.S. Pat. No. 4,416,777, disclose a material for separating leukocytes from a leukocyte-containing suspension which comprises a fibrous material having a surface layer coated on the fibrous material which can be dissolved by degrees in water.
None of the above-noted patents provides a reliable means for metering the rate of flow delay through a layer in order to retain a sample in contact with a reagent for a predetermined length of time, nor where the length of time can be varied depending on the incubation time required by the assay.
Alkyl ketene dimers have been used for many years as reactive alkaline sizing agents in the paper industry. Industrially, alkyl ketene dimers are added to the "wet end" of the papermaking process, that is, the slurry of bleached pulp in water that is at the very start of the papermaking process. Aqueous alkyl ketene dimer emulsions, which contain alkyl ketene dimer and additives such as defoamers and biocides, have been developed and marketed for this application. Wet-end addition allows for thorough deposition of the alkyl ketene dimer throughout the cellulose fiber, and is a less costly process than post-treatment of the fibers.
Gupta et al., in U.S. Pat. No. 5,071,675, disclose sizing cellulose fibers by applying a solution of alkyl ketene dimer in ethanol to cellulose fibers. Kortmann et al., in U.S. Pat. No. 5,028,236, disclose treating wool and synthetic polyamide materials with ketene dimers. Dereser, in U.S. Pat. No. 4,241,136, discloses a cationic size composition for glass fibers based upon a cationic film-forming polymer which also includes an alkylketene dimer. The coated glass fibers are then coated with an anionic size composition containing an anionic film-forming polymer whereby the cationic and anionic polymers react to form a thin film on the glass fibers.
No admission is made that any of the patents cited above constitutes prior art.